1. Field of the Invention
The invention relates to a new paradigm of disease centering around the metabolic pathways of S-adenosyl-L-methionine (SAM), the intermediates of these pathways and other metabolic pathways influenced by the SAM pathways. Specifically, the invention relates to analyzing and regulating SAM pathways that exist in association with a disease or condition including cancer and a number of diseases or conditions connected with degeneration and aging. More specifically, the invention concerns designing analytical, diagnostic and therapeutic protocols and agents for such disease states and conditions through recognition of the central role of SAM and its metabolic pathways in controlling cell metabolism, cell growth and intercellular communication.
2. Description of the Background Art
It is commonly believed that an understanding of cellular metabolism and function, as well as the nature of biological degeneration and the creation of disease conditions, can be achieved by ascertaining the genetic information contained in eukaryotic cells and understanding how this genetic information is transcribed and translated into proteins which then control chemical conversions within the cell. The present conceptual frame work considers DNA as the core of life. Within this framework, the function of proteins is commonly assumed to be regulated in large part by phosphorylation and dephosphorylation of relevant proteins at appropriate times.
The present invention was made in response to the absence of a greater unifying relationship between small molecule biochemistry and the macromolecules RNA, DNA and protein. Present-day molecular biology is focused completely on macromolecules and has provided essentially no connection with the small molecules which carry out the chemical reactions of life. In fact, the study of small molecules in the biological and biochemical sciences has not been in vogue for the last 20 years.
The present inventors have recognized that the focus of the present paradigm for metabolic management are at best very incomplete. Such a recognition demands a xe2x80x9cparadigm shiftxe2x80x9d of the sort described by Thomas Kuhn in The Structure of Scientific Revolutions (2nd ed., University of Chicago Press (1970)). The prevailing view will change completely when a single paradigm shift is made which defines the conceptual basis for the present invention. This paradigm shift is to the view of the present invention, that life is regulated by S-adenosyl-L-methionine (SAM) through at least eight xe2x80x9cdonor/cleavagexe2x80x9d pathways. In addition, as conceived by the present inventors, significant regulation occurs at the RNA level as opposed to the DNA level.
It is proposed here that our present state of knowledge and interpretation of disease has evolved into two classes which are termed by the present inventors: (1) xe2x80x9cexogenous diseasexe2x80x9d, for example that caused by viruses, bacteria and organisms exogenous to the affected subject, and (2) xe2x80x9cendogenous diseasexe2x80x9d, such as cancer and arthritis, largely associated with internal changes in the affected subject and frequently associated with aging. At present, understanding of exogenous disease is greater due to our knowledge of the external and identifiable causative agents. This knowledge has provided diagnostic and therapeutic procedures which are quite effective against many pathological states, such as bacterial infections. Comparatively speaking, we understand surprisingly little about endogenous diseases. While an enormous body of facts surrounding most endogenous diseases exists, we unfortunately have minimal molecular understanding of their etiology. The reason for this is simple: we have been adhering to the wrong paradigm, a DNA-centered conceptual framework which does not include regulation through RNA or through the eight pathways of SAM.
With the new view of life in accordance with the paradigm shift described herein, the diseases of man can be understood from an entirely new perspective. Most importantly, and practically, from this novel view of life and biochemical processes, the present invention suggests ways to discover and implement new diagnostics and therapeutics for virtually every endogenous disease of man, as well as many exogenous diseases such as bacterial and viral infections.
The recognition of the centrality of the eight xe2x80x9cdonor/cleavagexe2x80x9d pathways of SAM by the present inventors and their application to disease will radically alter the existing paradigm since SAM merges the biochemistry of small molecules with that of macromolecules. In order to design methods to assess the overall metabolic state of a eukaryotic organism and to modulate this state in response to a disease or a dysfunctional condition, the central role of SAM in controlling a host of biochemical reactions must be recognized and employed.
A number of publications which describe SAM and SAM metabolism are delineated below.
Several U.S. patents describe SAM, its activity, and various metabolites and utilities thereof U.S. Pat. No. 4,369,177 discloses a stable composition of SAM which includes a salt of SAM in a pharmaceutically acceptable water-soluble salt of a bivalent or trivalent metal. SAM is said to have pharmaceutical effects in various disorders including adipohepatica, hyperlipemia, arteriosclerosis, depression, arthritis deformans, pains in some neurological manifestations and sleeplessness.
U.S. Pat. No. 4,956,173 discloses use of ademethionine (a common name for a SAM salt) for the preparation of pharmaceutical or cosmetic compositions for counteracting aging of the skin. Ademethionine is said to be a physiological molecule of virtually ubiquitous distribution in the tissues and liquids of the organism where it is involved in important biological processes as a donor of methyl groups in numerous transmethylation reactions and as a precursor of physiological sulfur compounds such as glutathione, cysteine, taurine and CoA. Levels of SAM are known to be high in children and adolescents whereas they are lower in adults and subsequently decrease in presenility and senility. SAM is the active principle of drugs used especially for the treatment of degenerative osteoarthropathy where it has an important role through its antiphlogistic and analgesic activity due to its intervention in the metabolism of arachidonic acid and prostaglandins. Ademethionine is also indicated in the treatment of depressive syndromes.
U.S. Pat. No. 4,764,603 discloses SAM salts with water-soluble polyanions such as polyphosphates, polyvinylsulfonates, sulfates or phosphates, polyacrylates, polystyrene sulfonates.
U.S. Pat. No. 5,073,546 discloses liposoluble salts of SAM with acyl derivatives of taurine. This reference lists a number of important biochemical functions of SAM and summarizes various pharmacologic effects of SAM in test models and the use in clinical pathologies.
U.S. Pat. No. 4,605,625 discloses production of S-adenosyl-L-homocysteine (SAH) by contacting adenosine with D-homocysteine in an aqueous medium in the presence of Pseudomonas cells having the ability to racemize D-homocysteine to D-L-homocysteine and in the presence of SAH hydrolase to synthesize SAH. SAH is said to be an important biologically active substance formed by a methyl group donating reaction in vivo involving SAM. SAH was said to be efficacious as a sedative and a sleep-inducing agent.
U.S. Pat. No. 4,562,149 discloses yeast cultures containing SAM in high concentrations and a process for producing SAM by cultivating the yeast in a liquid culture medium containing methionine.
U.S. Pat. No. 4,242,505 discloses stabilized SAM compositions a sulfuric acid equivalent and a nucleoside sulfate. U.S. Pat. No. 4,057,686 discloses stable sulfonic acid salt of SAM.
U.S. Pat. No. 4,028,183 discloses preparation of double salts of SAM by the action of the enzyme ATP-methionine-adenosyl-transferase on a mixture of ATP and methionine. U.S. Pat. No. 3,954,726 discloses double salts of SAM with sulfuric acid and p-toluene sulfonic acid.
U.S. Pat. No. 3,962,034 discloses production of SAM and methylthioadenosine by yeast cultured in media containing L-methionine.
U.S. Pat. No. 4,599,309 discloses treatment of yeast cells following cultivation to facilitate product recovery. Among the many substances recoverable from such cells are SAM.
U.S. Pat. No. 5,100,786 discloses a gene which provides cellular resistance to at least one methionine derivative and is capable of enhancing accumulation of SAM in a cell. Also disclosed are a plasmid containing this gene, a cell transformed with the plasmid and a process for producing SAM using the above cells in a large amount at low cost. SAM is said to participate in metabolism of fats, proteins, sugar chains and the like and to have effects in the therapy of excessive lipemia, arteriosclerosis, psychosis manifestations such as depression and neuropathic diseases, degenerative arthropathy, neuropathic pain, sleeplessness, brain damage, and the like. The gene described in this reference is referred to as the xe2x80x9cSAM genexe2x80x9d which enables cells to accumulate SAM in large amounts though the gene also provides the cell with ethionine resistance.
U.S. Pat. No. 5,132,291 discloses antiviral purine nucleosides, analogs and prodrugs and methods of enhancing the antiviral activity of AZT. Some of the purine nucleoside analogs inhibit SAH hydrolase, an enzyme which converts SAH to adenosine and homocysteine. Inhibition of this enzyme causes a build-up of SAH, which in turn inhibits SAM-mediated methylation and the conversion of SAM to SAH. This latter inhibition is said to destabilize viral mRNA which is normally stabilized in part by methylation at the 5xe2x80x2-terminus to form the cap structure found in mammalian mRNA.
U.S. Pat. No. 4,376,116 discloses compounds which inhibit polyamine biosynthesis and cites U.S. Pat. Nos. 3,954,726 and 4,028,183 as describing the preparation of stable salts of SAM which is the parent compound of decarboxylated SAM (dcSAM). Such polyamine synthesis inhibitors are said to be useful as antiparasitic agents and in the treatment of cancer and cystic fibrosis.
U.S. Pat. No. 5,087,417 states that the biosynthetic path leading to ethylene formation begins with conversion of methionine to SAM, SAM to 1-amino-cyclopropane-1-carboxylic acid (ACC), and thence to ethylene. This reference describes the isolation and identification of the compounds which inhibit senescence in perishable plant tissue from petals of senescing carnation flowers. The active compound, a glucose ester of ferulic acid, inhibits ACC-to-ethylene conversion, ethylene formation in vitro and lipoxygenase activity.
U.S. Pat. No. 4,275,150 discloses an assay for measurement of normetanephrine in biological systems of patients in particular for hypertension or for detection of pheochromocytoma. This method utilizes conversion of normetanephrine to its N-methylated (and tritiated) derivative, metanephrine, utilizing SAM as methyl donor
U.S. Pat. Nos. 5,264,355 5,198,358 5,149,701 discloses a new methylating enzyme from Streptomyces, which uses SAM as a methyl donor in methylating various FK 506-related agents. These immunosuppressants are said to be useful for treating autoimmune diseases, infectious diseases, graft rejection, reversible obstructive airway disease, inflammatory and hyperproliferative skin disease, cutaneous manifestations of immunologically mediated illness, male pattern alopecia and alopecia senilis.
None of the documents cited above provide any insight into the present invention nor do they lead a person of ordinary skill in the art to the present claims.
The invention provides a new and useful paradigm for understanding a number of disease states or unwanted conditions and for identifying suitable molecular targets for intervention to control or ameliorate such diseases or conditions. This approach allows assessment of the overall biochemical/metabolic status of an organism. The methods and compositions focus on the eight metabolic pathways involving, and ultimately controlled by, SAM.
Thus, in one aspect, the invention is directed to a method to identify a therapeutic composition or protocol which modulates metabolism of a SAM pathway in a subject having a disease or undesired condition associated with altered SAM metabolism. The intended composition or protocol identified here, which is useful in ameliorating the disease or condition, is one which has not been previously identified as being useful for treating the particular disease or condition. The method comprises a number of steps:
a) determining the presence of the disease or condition in the subject or a biological fluid thereof;
b) identifying one or more of the SAM pathways or a metabolite of the pathways which is abnormal in amount, activity or modification and/or association with other cellular proteins, in the disease or condition;
c) determining which of the abnormal pathways or metabolites should be increased or decreased in activity or amount in order to ameliorate the disease or condition, thereby generating a first data set of target pathways or metabolites;
d) identifying a therapeutic composition or protocol which stimulates or suppresses the target pathway or increases or decreases the amount of the target metabolite,
thereby identifying the therapeutic composition or protocol useful in ameliorating the disease or condition.
In another embodiment, this method further comprises, after step (b) above, the step of prioritizing the pathways or metabolites of the first data set in order of deviation from normal to obtain a second data set of one or more target pathways and/or metabolites which are most abnormal, and wherein step (d) comprises identifying a therapeutic composition or protocol which stimulates or suppresses the second data set target pathway or increases or decreases the amount of the second data set target metabolite.
In the above methods, the first or second data set may be converted into digital computer-readable form for storage, access, manipulation, analysis and educational use. The results of any determination or identification made in any of the steps of the various methods disclosed herein may likewise be converted into digital computer-readable form.
In the above method, the identifying step (b) may be performed by subjecting a biological fluid obtained from the subject to (i) direct measurement of the concentration of one or more of the metabolites or the activity of an enzyme; or
(ii) affinity chromatography with immobilized affinity ligands which bind to SAM metabolites or macromolecules involved in a SAM pathway. Such an affinity-based identification (xe2x80x9chardwiringxe2x80x9d) approach may not identify all the potential SAM paradigm-based targets. For example, proteins, nucleic acids, lipids, sugars, and other small molecules may be modified by SAM or SAM pathway intermediates. These aberrant modifications in diseased states may not always be detected by the above affinity-based method. In these instances an additional approach (iii) termed xe2x80x9clabelingxe2x80x9d is preferred, comprising the following steps:
(a) obtaining a biological fluid from a subject having the disease or condition;
(b) contacting the fluid representing the disease state, which may be in the form of isolated enzyme(s), proteins, nucleic acids, a cell-free system, cultured cells, or an in vivo model of the disease state, with detectably labeled, preferably radiolabeled, SAM or a SAM pathway intermediate, and thereby allowing labeling of SAM pathway intermediates or of molecules to which the radiolabel is donated from the labeled SAM pathway precursor (intermediate);
(c) isolating any labeled components from the fluid;
(d) identifying any such labeled components;
(e) performing steps (a) to (d) with a biological fluid from a normal subject or a subject not having the disease or conditions or from another appropriate control fluid: and
(f) comparing the results of step (d) with the results in step (e), thereby identifying the change in the activity or amount of the components.
Alternatively, the labeling technique can be altered to examine kinetic vs. thermodynamic differences between the diseased and non-diseased states. For such an approach the steps (a) and (b) are carried out as above followed by:
(c) administering a large pulse of a radiolabeled metabolite;
(d) after an appropriate interval, stopping the reaction or adding unlabeled metabolite;
(e) isolating and identifying labeled components (labeled either under steady-state or pulse-chase conditions) in the sample;
(f) performing steps (a) to (e) with a biological fluid from a normal subject or a subject not having the disease or conditions or from another appropriate control fluid: and
(g) comparing the results of step (d) with the results in step (f), thereby identifying the change in the activity or amount of the components.
Yet another alternative involves assaying growth conditions of the biological fluid containing cells by adding a combination of SAM metabolites (pathway intermediates) and/or an inhibitor thereof in a combinatorial manner.
Another method provided herein is directed to evaluating a test compound which is a small molecule which is a SAM pathway component or a component which influences a SAM pathway, and which molecule has a molecular weight preferably below 5 kDa, for its utility in a therapeutic composition or protocol for treating disease or condition associated with altered SAM metabolism. In one embodiment of this method, cells associated with the disease or condition (obtained from a subject or from a cultured cell line) are grown in a selected medium which includes one or several concentrations of the small molecule (or a combination of small molecules) being evaluated, preferably in a combinatorial manner. The impact of the small molecule on a target cellular process characteristic of the disease or condition is determined by measuring the target cellular process using any analytical method well-known in the art. Examples of target processes include: transport of molecules across the cell membrane, intracellular localization of molecules in organelles or compartments, intercellular and intracellular signalling pathways, metabolic processes, and the like. The results obtained from this determination are compared with the results obtained using the same cells grown without the added test compound or compounds. Alternatively, or additionally, the results are compared to the results obtained with normal cells (not associated with the disease or condition) grown in the presence of the same concentrations of the same test compound or compounds. Such a comparison will identify compounds which are capable of altering the target cellular process and are thereby useful in a therapeutic composition or protocol for the disease or condition. This method is particularly useful with cancer cells grown in culture.
In a different embodiment of the above method, rather than evaluating a compound""s action on cells in vitro, an animal model of the disease or condition is administered the test compound or compounds in vivo. The target cellular process analyzed by obtaining a biological fluid, preferably including cells, from the animal and analyzing it as above. A comparison is made with a normal animal counterpart of the disease model. This method is particularly amenable to evaluating a compound for its utility in diabetes, various autoimmune diseases, or any genetically based metabolic disorder for which an animal model is known or becomes known in the future.
In all the above methods the disease or condition is preferably selected from the group consisting of a wound, cancer, multiple sclerosis, Alzheimer""s disease, Parkinson""s disease, depression and other imbalances of mental stability, atherosclerosis, cystic fibrosis, diabetes, obesity, Crohn""s disease, and altered circadian rhythmicity. The invention can also be practiced with any other disease or condition not specifically listed or exemplified herein. Such diseases are described, for example, in Scriver et al (Eds), THE METABOLIC BASIS OF INHERITED DISEASE, McGraw Hill Information Services Company, New York. Volumes I and II, 1989; Graig et al., MODERN PHARMACOLOGY, 4th Edition, Little Brown and Company, Boston, 1994). Other categories of diseases amenable to the methods of the present invention include arthritis, psoriasis and other skin diseases, autoimmune diseases, allergies, hypertension, anxiety disorders, schizophrenia and other psychoses, osteoporosis, muscular dystrophy, amyotrophic lateral sclerosis and circadian rhythm-related conditions. The methods and approaches disclosed herein are in fact applicable to an exogenous or endogenous disease or metabolic alteration in any living organism, ranging from unicellular organisms to plants and higher animals. Preferred subjects for the present methods are mammals. Preferred mammals are animals of agricultural importance. Most preferred subjects are humans.
When the condition is a wound, the target metabolites preferably comprise one or more of SAM or a SAM derivative, methionine, adenosine, cysteine, homocysteine, cystathionine, choline, ethylene, biotin, biotin analogues, ACC, polyamines, queuosine, queuine and nicotinamide. For cancer the target pathways or metabolites preferably comprise one or more of methylation products of RNA, methylation products of DNA, methylation products of protein, methylation products of a small molecule, queuosine, queuin, wye-base incorporation into tRNA or effects promoted by queuine, SAM synthesis, folate and vitamin B12 transfer of methyl groups, methionine synthesis, methylthioadenosine or its catabolic products, homoserine lactone, 5-deoxyadenosine, polyamine synthesis and catabolism, ethylene synthesis, biotin levels, hypusine synthesis on eIF-5A (formerly termed eIF4D), diphthamide synthesis on EF-2 and salvage of methylthioadenosine (specifically, adenine phosphoribosyl transferase, APRT) and the enzymes which convert the ribose moiety to methionine. When the disease is multiple sclerosis, the target pathways or metabolites preferably comprise one or more of: levels of SAM, S-adenosyl homocysteine, folate and vitamin B12 levels in serum and cerebrospinal fluid, methyl transferase activity, myelin basic protein methylation and phosphatidylcholine levels. For Alzheimer""s disease and the target pathways or metabolites preferably comprise one or more of methylation levels, SAM, biotin, polyamines, Ca2+/calmodulin methylation and levels, folate, vitamin B12, ubiquinone and alternative splicing of mRNA for xcex2-amyloid protein. For Parkinson""s disease, the target pathways or metabolites preferably comprise one or more of polyamines, nonspecific N-methylase, acetyl-L-carnitine, Ca2+/calmodulin-dependent protein kinase II, lysolecithin, sphingomyelin, SAM and vitamin B12. When the disease is atherosclerosis, the target pathways or metabolites preferably comprise one more of methylation levels, homocysteine and its catabolites, polyamines, acetyl-L-carnitine, calmodulin, and essential phospholipids. For cystic fibrosis the target pathways or metabolites preferably comprise one or more of calmodulin, polyamine levels, polyunsaturated fatty acids, total saturated and monounsaturated fatty acids and the cystic fibrosis transmembrane conductance regulator. When the condition is obesity the target pathways or metabolites preferably comprise one or more of methylation levels, serotonin, calmodulin, carnitine and ubiquinone.
The present invention is further directed to a method of treating a subject having a disease or undesired condition associated with altered SAM metabolism, comprising administering a therapeutic composition or protocol identified by the method described above to the subject, with the proviso that the therapeutic composition or protocol is one which has not been previously identified as being useful for treating the disease or condition.
The present invention is directed to a therapeutic composition or protocol identified by the above method, with the above proviso. The therapeutic composition or protocol is preferably one which is used to treat a disease or condition is selected from the group consisting of (but not limited to) a wound, cancer (including leukemias and lymphomas), multiple sclerosis, Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, depression or other mental instabilities, atherosclerosis, cystic fibrosis, diabetes, obesity, arthritis, autoimmune diseases (such as Crohn""s disease), arthritis, psoriasis, allergies, hypertension, osteoporosis, cystic fibrosis, and altered circadian rhythmicity.
In one embodiment, the above therapeutic composition is for treating a wound and comprises adenosine, methionine, 1-amino-cyclopropane-1-carboxylic acid, biotin and nicotinamide in amounts effective for treating the wound.
Also provided is a pharmaceutical composition useful in treating a disease or undesired condition in a subject, which comprises an effective amount of the above therapeutic composition and a pharmaceutically acceptable excipient, optionally in combination with one or more additional agents useful for treating the disease or condition. A preferred pharmaceutical composition for treating a wound comprises adenosine, methionine, 1-amino-cyclopropane-1-carboxylic acid, biotin and nicotinamide.
The invention provides a method to treat a disease or undesired condition associated with altered SAM metabolism in a subject, which comprises administering to the subject a therapeutic composition or protocol or a pharmaceutical composition, as above.
The present invention further is directed to a method to characterize a disease or undesired condition, or to characterize the progress of the disease or condition, which disease or condition is associated with altered SAM metabolism, the method comprising:
(a) identifying one or more metabolic pathways associated with the disease or condition which metabolic pathway is a SAM pathway or is influenced by a SAM pathway or a metabolite of a SAM pathway;
(b) determining which of the one or more metabolic pathways identified in (a) is altered from normal in the presence of the disease or condition and which pathway changes in response to the progression thereof;
(c) measuring the activity, amount or association with other proteins, of selected enzymes or metabolites of the one or more pathways determined in step (b) as a function of time after onset of, or diagnosis of, the disease or condition, to obtain a metabolic data set;
(d) recording clinical manifestations of the progression of the disease or condition to obtain a clinical data set
(e) comparing the metabolic data set with the clinical data set to obtain a combined data set,
wherein the metabolic, clinical or combined data sets characterize the disease or condition, or characterize the progression thereof Any of the above data sets may be converted to digital computer-readable form.
Also provided is an electronically retrievable profile characterizing a disease or undesired condition or the progression thereof, which disease or condition is associated with altered SAM metabolism, which profile is obtained by the above method.
Also included is a method for identifying or measuring a change in the activity, amount or association with another protein, of (i) a component of a SAM pathway or (ii) a component of a metabolic pathway which is influenced by a SAM pathway, in a subject having a disease or undesired condition associated with altered SAM metabolism, which method comprises:
(a) obtaining a biological fluid from a subject having the disease or condition;
(b) contacting the fluid with an affinity matrix to which is immobilized at least one first affinity ligand, which ligand (or ligands) binds to an enzyme or metabolite of a SAM pathway, and allowing material in the fluid to bind to the ligand;
(c) removing any material of the fluid not bound to the ligand;
(d) eluting material bound to the ligand from the affinity matrix to produce a first eluate, in a single fraction or as sequential fractions eluted with increasing salt concentrations or with different competitive ligands;
(e) identifying the presence or measuring the amount of any previously bound and subsequently eluted material in the first eluate;
(f) performing steps (a)-(e) with a biological fluid from a normal subject not having the disease or condition; and
(g) comparing the results of step (e) with the results of step (f),
thereby identifying or measuring the change in the activity, amount or association of the components.
The above method may comprise the additional steps, prior to step (e), of
(h) contacting the first eluate of step (d) with a second affinity matrix to which is affixed at least one second affinity ligand different from the first affinity ligand, which second ligand binds an enzyme or metabolite of a SAM pathway, and allowing material in the eluate to bind to the second ligand;
(i) removing any material of the first eluate not bound to the second ligand of step (h);
(j) eluting any material bound to the second ligand from the affinity support, thereby producing a second eluate;
(k) identifying the presence or measuring the amount of any previously bound and subsequently eluted material in the second eluate;
(l) performing steps (a)-(e) and (h)-(k) with a biological fluid from a normal subject not having the disease or condition; and
(m) comparing the results of step (l) with the results of step (k),
thereby identifying or measuring the change in the activity, amount or association, of the components.
Also provide herein is a method for identifying or measuring a change in the activity or amount of (i) a component of a SAM pathway or (ii) a component of a metabolic pathway which is influenced by a SAM pathway, in a subject having a disease or undesired condition associated with altered SAM metabolism, which method comprises:
(a) obtaining a biological fluid from a subject having the disease or condition;
(b) contacting the fluid with a detectably labeled compound selected from the group consisting of SAM, a SAM pathway metabolite other than SAM and a molecule which indirectly affects a SAM pathway, thereby allowing the detectable label to be donated to any other molecule in a SAM pathway or a pathway influenced by a SAM pathway, thereby resulting in labeled products in the fluid;
(c) identifying the presence of, or measuring the amount of, any of the labeled products;
(d) performing steps (a)-(c) with a biological fluid from a normal subject not having the disease or condition; and
(e) comparing the results of step (e) with the results of step (f),
thereby identifying or measuring the change in the activity, amount or association, of the components.
In the above method, the subject may be a non-human animal model of the disease or condition.
Another method involves determining the concentration of intermediates of the SAM pathways in biological fluids including any types of clinically relevant samples. This method, which may be used alone or in combination with the methods described above, includes the steps of:
(a) obtaining a biological fluid from a subject having the disease or condition;
(b) determining the concentration of SAM and/or SAM pathway intermediates and/or molecules influenced by SAM and/or SAM pathway intermediates. These concentrations can be determine by any or a number of methods known in the art of which HPLC is preferred. The metabolites or intermediates may include, SAM, methionine, SAH, 5xe2x80x2-deoxyadenosine, 5xe2x80x2-methylthioadenosine, dcSAM, putrescine, spermine, spermidine, derivatized forms of these polyamines such as acetyl spermine, acetyl spermidine or glutathione-conjugated spermidine, homocysteine, cAMP, cGMP, adenosine, inosine, homoserine lactone, queuine, queuosine, wye-base, etc.
(c) performing step b) with a biological fluid from a normal subject not having the disease or conditions: and
(d) comparing the results of step b) with the results in step c) thereby identifying the change in the activity or amount of the components.
From the data generated above with these methods, both pharmaceuticals and diagnostic products can be developed. In some instances in a diseased state, abnormal metabolism may be suspected which involves binding of proteins or enzymes to the cytoskeleton. In such a case, a preferred approach employs cytoskeletal molecules, e.g., actin or tubulin, immobilized to an affinity matrix. This affinity matrix is then used to examine a biological fluid from a diseased and non-diseased state for interaction of SAM and SAM-intermediates with the affinity matrix or with cell components previously bound to the affinity matrix. This information is then used to developed pharmaceuticals or diagnostic products.
Diagnostic Products
The presence or absence of any component (SAM pathway or intermediate) in the disease state or, alternatively, a large difference in any component between the normal and disease state, will lead to the development of a diagnostic assay by methods known in the art. As an example an antibody can be developed against the targeted SAM pathway component by standard procedures known in the art. This antibody can be used in an immunoassay, preferably an solution-based enzyme inmmunoassay (EIA) which may be measured by colorimetry, fluorescence or chemiluminescence. Alternatively, an in situ assays or a dipstick-based assay may be preferred.
In situations wherein the concentrations of the components being measured is at an appropriate detectable level, direct assays may be used, for example colorimetric, fluorometric chemiluminescent, HPLC, gas chromatographic, etc.
Therapeutic Products
When the concentration of one or more SAM pathway intermediates is found to be aberrant in a disease state, the condition is treated by administering the appropriate SAM intermediate or intermediates to the subject. Alternatively, the condition is treated by inhibition of stimulation of the particular SAM pathway or pathways. For example, the SAM/SAH ratio can be decreased by the use of SAH hydrolase inhibitors (Wolfe et al., J. Med. Chem. 34:1521-1530 (1991)) or adenosine deaminase inhibitors. Similarly, the use of the polyamine antagonist DL-xcex1-difluoromethylornithine, can increase SAM concentrations as much as 48-fold (Bacchi et al., In: Parasitic Protozoa and Polyamines in Inhibition of Polyamine Metabolism, McCann, P. P. et al., eds., Academic Press, New York, pp 317-344 (1987)).
Aberrant Binding of a Ligand to a Target Molecule in a Disease State
In some disease states, a ligand such as a protein, nucleic acid, lipid, sugar, or other molecule may bind abnormally to a target molecule. In these cases, displacement of the ligand from its target may improve the subject""s condition and ameliorate the disease. An assay, preferably in microtiter plate format, that measures the ligand having the undesired binding, is used according to procedures known in the art as a screening assay to develop active compounds. Particularly preferred is the use of a screening assay in combination with combinatorial drug discovery procedures (see for example, Desai et al., Drug Devel Res. 33:174-188 (1994); Jacobs et al., Trends in Biotech. 12:19-26 (1994)) to develop active compounds. Similarly a therapeutic approach may involve decreasing or increasing the activity of one or more enzymes. Thus, enzyme inhibitors or activators may be developed with the use of the appropriate screening assay and combinatorial drug discovery approach.